*Target (protein/gene name): Aldehyde Reductase *NCBI Gene # or RefSeq#: 24070 *Protein ID (NP or XP #) or Wolbachia#: LmjF31.2880 *Organism (including strain): Leishmania major Etiologic Risk Group (see link below): Risk Group 2 - Parasitic Agent *Background/Disease Information (sort of like the Intro to your Mini Research Write up): Leishmania major is a parasite (protozoan) that causes Leishmaniasis, a disease transmitted by the bite of sand flies. Leishmaniasis symptoms include skin sores that appear weeks or months after the individual has been bitten by sand flies. There are four main types of Leishmaniasis, visceral, cutaneous, diffuse cutaneous, and mucocutaneous Leishmaniasis. Regions experiencing the most cases of Leishmaniasis include the Middle East, and Asia due to the increased presence of sand flies. Leishmania major relies upon enzymes and lysozyme inhibitors to reproduce in sand fly hosts. Currently there are no vaccines in use, however efforts to develop them have been increased in the 21st century. Essentiality of this protein:
Gene/Ortholog: mtu3021 (OG4_10049); Phenotype: essential; Source study: nmpdr Complex of proteins?: No. Druggable Target: 0.5 druggability index (Yes). *EC#: 1.1.1.21 Link to BRENDA EC# page: http://brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.21
-- Show screenshot of BRENDA enzyme mechanism schematic: Unable to find mechanism.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Aldehyde reductase (E.C. 1.1.1.2) has been purified to apparent homogeneity from rat lens and its properties have been compared to those of both rat lens aldose reductase and rat kidney aldehyde reductase. The enzyme assay needed is
"Amicon Matrex Gel Orange A" and relies upon NADPH activity at 340nm. -- link to Sigma (or other company) page for assay or assay reagents (substrates) http://www.sigmaaldrich.com/catalog/product/sial/13767?lang=en®ion=US -- link (or citation) to paper that contains assay information http://www.sciencedirect.com/science/article/pii/105687279390043X -- List cost and quantity of substrate reagents and supplier NADPH: $66/25mg Structure Available (PDB or Homology model) -- PDB # or closest PDB entry if using homology model: -- For Homology Model option: ---- Show pairwise alignment of your BLASTP search in NCBI against the PDB ---- Query Coverage: ---- Max % Identities: ---- % Positives: ---- Chain used for homology:
Current Inhibitors: A broad group of structurally diverse aldose reductase inhibitors including flavonoids, carboxylic acids and hydantoins, have been tested (aldehyde reductase is inhibited by a number of aldose reductase inhibitors). Expression Information (has it been expressed in bacterial cells): No, it is a eukaryotic enzyme. Purification Method: Purification is accomplished by ammonium sulfate fractionation, Sephadex G-75 chromatography, affinity chromatography on Amicon Matrex Gel Orange A and chromatofocusing.
Image of protein (PyMol with features delineated and shown separately): Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MAARRVVRLPDGTAAPALGQGVWMMGETPENRTRELAALRAGMEAGMTLIDTAEMYGNGRSERLVAEAIKETPRERLFIVSKVLPTNASRAAIFRSCDASLQNIGTDYLDLYLLHWRGRVPLSETVTCMEELVKSGKIRRWGVSNFDVDDMKELWRVPGGDKCAVNQVLYHLGSRGIEYDLLPWLREHKVPVMAYCPIAQAGELKSELYKSTVVRSVAARHNATVTQVLLAFVLRSGHVIAIPRSSNPAHTEENAAADSIELTEADMAQLDAAFPAPKRKTHLDIV *length of your protein in Amino Acids: 286 amino acids in Aldehyde Reductase. Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 31621 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 38180 M^-1 cm^-1 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. -N-terminus on inside, with 1 strong transmembrane helix. Unlikely to be transmembrane.
*GC% Content for gene: 62% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized): Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene # or RefSeq#: 24070
*Protein ID (NP or XP #) or Wolbachia#: LmjF31.2880
*Organism (including strain): Leishmania major
Etiologic Risk Group (see link below): Risk Group 2 - Parasitic Agent
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Leishmania major is a parasite (protozoan) that causes Leishmaniasis, a disease transmitted by the bite of sand flies. Leishmaniasis symptoms include skin sores that appear weeks or months after the individual has been bitten by sand flies. There are four main types of Leishmaniasis, visceral, cutaneous, diffuse cutaneous, and mucocutaneous Leishmaniasis. Regions experiencing the most cases of Leishmaniasis include the Middle East, and Asia due to the increased presence of sand flies. Leishmania major relies upon enzymes and lysozyme inhibitors to reproduce in sand fly hosts. Currently there are no vaccines in use, however efforts to develop them have been increased in the 21st century.
Essentiality of this protein:
Gene/Ortholog: mtu3021 (OG4_10049); Phenotype: essential; Source study: nmpdr
Complex of proteins?: No.
Druggable Target: 0.5 druggability index (Yes).
*EC#: 1.1.1.21
Link to BRENDA EC# page:
http://brenda-enzymes.org/php/result_flat.php4?ecno=1.1.1.21
-- Show screenshot of BRENDA enzyme mechanism schematic: Unable to find mechanism.
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Aldehyde reductase (E.C. 1.1.1.2) has been purified to apparent homogeneity from rat lens and its properties have been compared to those of both rat lens aldose reductase and rat kidney aldehyde reductase. The enzyme assay needed is
"Amicon Matrex Gel Orange A" and relies upon NADPH activity at 340nm.-- link to Sigma (or other company) page for assay or assay reagents (substrates)
http://www.sigmaaldrich.com/catalog/product/sial/13767?lang=en®ion=US
-- link (or citation) to paper that contains assay information
http://www.sciencedirect.com/science/article/pii/105687279390043X
-- List cost and quantity of substrate reagents and supplier
NADPH: $66/25mg
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage:
---- Max % Identities:
---- % Positives:
---- Chain used for homology:
Current Inhibitors:
A broad group of structurally diverse aldose reductase inhibitors including flavonoids, carboxylic acids and hydantoins, have been tested (aldehyde reductase is inhibited by a number of aldose reductase inhibitors).
Expression Information (has it been expressed in bacterial cells): No, it is a eukaryotic enzyme.
Purification Method: Purification is accomplished by ammonium sulfate fractionation, Sephadex G-75 chromatography, affinity chromatography on Amicon Matrex Gel Orange A and chromatofocusing.
Image of protein (PyMol with features delineated and shown separately):
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MAARRVVRLPDGTAAPALGQGVWMMGETPENRTRELAALRAGMEAGMTLIDTAEMYGNGRSERLVAEAIKETPRERLFIVSKVLPTNASRAAIFRSCDASLQNIGTDYLDLYLLHWRGRVPLSETVTCMEELVKSGKIRRWGVSNFDVDDMKELWRVPGGDKCAVNQVLYHLGSRGIEYDLLPWLREHKVPVMAYCPIAQAGELKSELYKSTVVRSVAARHNATVTQVLLAFVLRSGHVIAIPRSSNPAHTEENAAADSIELTEADMAQLDAAFPAPKRKTHLDIV
*length of your protein in Amino Acids: 286 amino acids in Aldehyde Reductase.
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite: 31621 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 38180 M^-1 cm^-1
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
-N-terminus on inside, with 1 strong transmembrane helix. Unlikely to be transmembrane.
*CDS Gene Sequence (paste as text only):
*GC% Content for gene: 62%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Resources: